Neurons transmit information via electrical signals. During the previous project period, the molecular components of voltage-sensitive sodium channels which are responsible for generation of action potentials in neurons were identified, purified and functionally reconstitiuted in purified form. The propose studies will continue this work. The subunits of the purified sodium channel will be preparatively separated and biochemically characterized. The molecular weights of the subunits and the sodium channel complex will be rigorously determined. The functional roles of the individual subnunits will be examined. The topology of the purified sodium channel and its subunits in reconstituted phospholipid vesicles will be determined and the components involved in channel inactivation and in binding of saxitoxin and terodotoxin will be identified by chemical modification. The role of specific phospholipids in the reconstitution of sodium channel function from purified components will be analyzed using both biochemical and electrophysiological recording methods. Modulation of channel function by the phosphorylation of specific sites by different protein kinases in situ and in purified and reconstituted vesicles will be studied. Molecular genetic methods will be developed to clone cDNA encoding the sodium channel subunits and the functional expression of mRNA and cloned genes will be studied by microinjection and electrophysiological recording from mammalian cells. The sodium channel will be localized by immunocytochemical methods in identified classes of central neurons. These experiments will define the structure/function relationships of the sodium channel as well as its modulation and localization in central neurons. These studies will provide essential information to understanding the molecular basis of electrical excitability.